PROJECT SUMMARY/ABSTRACT A long history of developmental biology research into avian and mammalian digit formation has defined the cellular and molecular underpinnings of the digit separation process. At a cellular level, apoptosis of the interdigital mesenchyme, as well as differential digit and interdigital growth, mediate digit separation. The interdigital apoptosis is initiated by decreasing FGF8 expression in the overlying epidermis which triggers a cascade of molecular changes in the interdigital mesenchyme, ultimately resulting in apoptosis and regression of the interdigital tissue. A failure of this process is thought to underlie a number of congenital abnormalities in humans with fused digits, a condition observed in 3-10 per 10 thousand births. Through our work on Grhl3-/- mice, we discovered an entirely new epithelial-based mechanism for digit separation. Our data suggest that while interdigital apoptosis is required for digit separation it is not sufficient. We identified the formation of a small multilayered epithelial tongue structure (referred to as the interdigital epithelial tongue; IET) proximally at the separating digit junctions. For normal digit formation, the epithelia within this structure needs to undergo bifurcation concomitant with the interdigital mesenchymal cell death. The hypotheses in this application are: 1) that as the interdigital juncture epithelium moves proximally and from the dorsal and ventral surfaces in response to interdigital mesenchyme cell death, a multilayered epithelial tongue structure forms at its leading edge; and 2) that a GRHL3-controlled pathway is required for bifurcation of the interdigital epithelial tongue to allow digit separation. We propose two Aims to test these hypotheses. In Aim 1, we will characterize the cellular mechanisms responsible for movement and bifurcation of the interdigital epithelial tongue. In Aim 2, we will define a GRHL3-regulated molecular pathway responsible for bifurcation of the interdigital epithelial tongue. These studies are significant and innovative because they define a previously unrecognized cellular and molecular pathway required for normal digit separation.